Many drugs suffer from unfavorable pharmacokinetic parameters that limit their effectiveness. Rapid clearance of such drugs from physiological compartments, either via metabolism or excretion, results in short lifetimes and reduced exposure to targets. For example, the therapeutic potential of peptide- and protein-based drugs is enormous, yet peptide- and protein-based drugs often suffer rapid systemic clearance due to metabolic instability and renal clearance. Similarly, nucleic acids such as antisense DNA and small interfering RNAs (siRNAs) have great therapeutic potential, yet suffer from metabolic instability and cell impermeability. Finally, many small organic molecules also suffer from rapid clearance that limits their therapeutic effectiveness.
It would thus be highly desirable to have methods to prolong the half-life in the systemic circulation and/or other physiological compartments and improve the availability and cell uptake of small molecule, peptide-, protein-, and nucleic acid-based therapeutics in order to provide improved drug- and gene-based therapies in the treatment of disease.
One method for increasing the physiological half-life of drugs is to increase their hydrodynamic size by attaching them to macromolecules. Removal of large molecules, for example high-molecular weight proteins, antibodies, polymers, poly(ethyleneglycol) (PEG), from the systemic circulation can be extremely slow. Metabolism of PEG having mw>5000 is insignificant, and both glomerular filtration and biliary excretion of PEG having mw ˜50 kDa is minimally effective. For example, the plasma half-lives in rates or mice of several PEG-superoxide dismutase (PEG-SOD) conjugates have been reported to vary from 1.5 hours for a conjugate using PEG of molecular weight 1900 to 36 hours for a conjugate using PEG of molecular weight 72,000, while the half-life of unconjugated SOD was 0.08 hour. See Veronese, “Peptide and protein PEGylation: a review of problems and solutions,” Biomaterials (2001) 22:405-417. Monoclonal antibody and serum albumin likewise have very long resident times in the systemic and other physiological compartments, which leads to greatly extended systemic/compartmental half-lives of macromolecular conjugated drugs, largely dependent upon the molecular weight of the conjugate.
For peptide- and protein-based drugs, covalent attachment of polymer residues, for example PEG (known as “PEGylation”), has been effective at improving the pharmacokinetic parameters and can also mask the drug agent from metabolism and from the immune system, leading to reduced immunogenicity. PEGylation has resulted in such modified drugs as PEG-bovine adenosine deaminase for the treatment of X-linked severe combined immunogenicity syndrome (ADAGEN®, Enzon), PEG-alpha interferon for the treatment of hepatitis C (PEGASYS®, Hoffman-LaRoche; PEG-Intron®, Schering-Plough/Enzon), PEG-L-asparaginase from the treatment of acute lymphoblastic leukemia (Oncaspar®, Enzon), PEG-recombinant human granulocyte colony stimulating factor for the treatment of neutropenia (Neulasta®, Amgen), PEG-anti-tumor necrosis factor alpha for the treatment of Crohn's disease (Cimzia®, Enzon), PEG-growth hormone receptor antagonist for the treatment of acromegaly (Somavert®, Pfizer), and PEG-anti-TNF Fab for rheumatoid arthritis (CD870, Pfizer).
PEGylation has also been shown to improve delivery of nucleic acids to cells. For example, US patent publication 2008/0064863 discloses double-stranded nucleic acids, one strand of which is covalently attached to a poly(ethyleneoxide) unit, in complex with a polycation for use in the delivery of nucleic acid drugs to cells. PCT publication WO2007/021142 discloses covalently PEGylated siRNA molecules. PEG-anti-VEGF aptamer has been approved for intraocular treatment of age-related macular degeneration (Macugen®, OSI/Pfizer).
PEGylation of small molecules has also been reported. EZN-2208, a PEG conjugate of SN-38, the active metabolite of irinotecan, has been shown to be active in preclinical tumor models. In this instance, PEGylation improves the solubility of the small molecule drug.
Covalent attachment of peptide and protein drugs to macromolecules other than PEG has been disclosed. For example, conjugates of various peptide drugs, such as thrombospondin-1 mimetic peptides, angiopoietin-2 antagonist, glucagon-like peptide-1 (GLP-1), and exendins, with a monoclonal antibody have been reported.
Covalent modification of peptides, proteins and small molecules with PEG, or other macromolecules, often causes deleterious loss of the biological activity of the parent drug, however. Thus, some recent activity has focused on development of reversible, or transient, PEGylation, in which the polymer chains are conjugated to the drug through a cleavable linker unit. The final PEGylated conjugate is of sufficient molecular size to have favorable systemic retention. Under physiological conditions, cleavage of the linker unit by enzyme or chemical action leads to release of a drug or prodrug that is rapidly converted to the active drug. Depending upon the rate of cleavage of the linker unit relative to the clearance rate of the prodrug or free drug, sufficient steady-state concentrations of active drug for biological activity may be realized using this approach.
Success has been reported using this approach, for example, using the immunotoxin SS1P reversibly-conjugated to PEG through lysine residues. See Filpula, et al., “Releasable PEGylation of Mesothelin Targeted Immunotoxin SS1P Achieves Single Dosage Complete regression of a Human Carcinoma in Mice,” Bioconjugate Chemistry (2007) 18:773-784. Whereas unmodified SS1P was eliminated from mouse plasma with a half-life of about 26 minutes, reversible PEGylation extended the half-life to 2.5-5 hours. Reversible PEGylation of atrial natriuretic peptide (having a plasma half-life of 2-5 minutes) has been shown to result in prolonged protracted effects on blood pressure in adrenaline-treated rats (Nesher, et al., Bioconjugate Chem (2008) 19:342-348).
Most methods for reversible PEGylated or other macromolecular conjugated drugs suffer potential drawbacks. For example, some require enzyme hydrolysis by serum proteases or esterases, others need a reducing environment to cleave a disulfide linker, and most release a “self-immolative” prodrug that undergoes spontaneous cleavage to the active drug and a small, potentially toxic alkylating agent. It would be beneficial to design versions of reversible PEGylation or macromolecular drug attachment that do not require and are unaffected by difficult-to-control entities such as enzymes, and redox environments.
U.S. Pat. No. 6,504,005 describes prodrug molecules that release active drug under physiological conditions by virtue of beta elimination department on pH. A specific embodiment of this approach is described in WO2004/089279. This approach, albeit limited in scope and examples, utilizing a spontaneous, first-order rate of cleavage of the drug from the PEG carrier that is initiated when the conjugate is exposed to physiological pH, is described in US Patent Publication 2006/0171920. A general strategy for providing macromolecule-drug conjugates having a variety of spontaneous, first-order release rates that are predictable and controllable under physiological conditions would provide a valuable therapeutic tool for the treatment of disease.